The formation of native disulfide bonds is at the core of oxidative protein folding. [1-4] In oxidizing environments, reduced proteins with multiple cysteine residues tend to oxidize rapidly and nonspecifically. To attain a proper three-dimensional fold, any non-native disulfide bonds must isomerize to the linkages found in the native protein. [5] In eukaryotic cells, this process is mediated by the enzyme protein disulfide isomerase (PDI; EC 5.3.4.1). [4,6-14]
Catalysis of disulfide-bond isomerization by PDI involves thiol-disulfide interchange chemistry (FIG. 1). [15] The mechanism commences with the nucleophilic attack by a thiolate on a non-native disulfide bond, generating a mixed-disulfide and a new substrate thiolate. This thiolate can then attack another non-native disulfide bond, inducing further rearrangements to achieve the stable native state (FIG. 1).
PDI is abundant in the endoplasmic reticulum (ER) of eukaryotic cells. The enzyme contains four domains: a, a′, b, and b′. [12] The a and a′ domains each contain one active-site CGHC motif—a pattern analogous to that in many other oxidoreductases, whereas the b and b′ domains appear to mediate substrate binding. [16, 17, 12] The physicochemical properties of its active-site make PDI an ideal catalyst for the reshuffling of disulfide bonds in misfolded proteins. In its catalytic mechanism (FIG. 1), the deprotonated thiolate of the N-terminal active-site cysteine (CGHC) initiates catalysis. [18] The amount of enzymic thiolate present is dependent on two factors. [19,20] One is the pKa of the active-site cysteine residue; the other is the reduction potential (E∘′) of the disulfide bond formed between the two active-site cysteine residues. In PDI, the cysteine pKa is 6.7, and the disulfide E∘′ is −0.18 V. [21, 22] Given the properties of the ER (pH 7.0; Esolution=−0.18 V), about 33% of PDI active sites will contain a reactive thiolate. [23, 24] Moreover, the high (less negative) reduction potential of PDI renders the protein as a weak disulfide-reducing agent, ensuring that ample time is available for the catalyst to rearrange all of the disulfide bonds before reducing its protein substrate to “escape” (FIG. 1). If necessary, the second active-site cysteine residue can engage to rescue the enzyme from non-productive mixed-disulfide intermediates. [7, 25, 26]
Efficient oxidative protein folding requires a redox environment that supports both thiol oxidation and disulfide-bond isomerization. In vitro and in cellulo, this environment can be provided by a redox buffer consisting of reduced and oxidized glutathione. For example, the oxidative folding of a common model protein, bovine pancreatic ribonuclease (RNase A)[27, 84], occurs readily in the presence of 1 mM glutathione (GSH) and 0.2 mM oxidized glutathione (GSSG). [28] Adding PDI accelerates the process, but the large-scale use of PDI as a catalyst for folding proteins in vitro is impractical due to its high cost and conformational instability, and the complexity imposed by its separation from a substrate protein. Accordingly, the development of small-molecule PDI mimics has become a high priority.
To date, most PDI mimics have been designed to replicate the physicochemical properties of the CGHC active site with low thiol pKa and high disulfide E∘′. [29] For example, BMC, (±)-trans-1,2-bis(mercaptoacetamido)cyclohexane (FIG. 2), a small molecule that catalyzes the formation of native disulfide bonds in proteins, both in vitro and in vivo, has been reported. [30] U.S. Pat. No. 5,910,435 reports that organic dithiols, exemplified by BMC, having a pKa of less than about 8.0 and a standard reduction potential (EO′) of greater than about −0.25 volts, are capable of catalyzing the formation of proper disulfide bonds in proteins, in the complete absence of PDI, both in vivo and in vitro. This patent also reports that BMC is capable both in vivo and in vitro of catalyzing the formation of the proper biologically active form of eukaryotic proteins produced in non-eukaryotic systems.
Fox and coworkers screened 14 reagents for their ability to fold a variety of proteins, and concluded that BMC was the best of known small-molecule catalysts. [31] Though effective, BMC has shortcomings. For example, its low disulfide E∘′ renders the compound too reducing for optimal catalysis of disulfide-bond isomerization. Subsequently, various CXXC and CXC peptides, aromatic thiols, and selenium-based catalysts have been reported to exhibit some success. [32-43] In addition to non-optimal thiol pKa and disulfide E∘′ values, these organocatalysts failed to mimic a hallmark of enzymic catalysts—binding to the substrate. [44] The b and b′ domains of PDI have an exposed hydrophobic patch. These patches unite to form a continuous hydrophobic surface between the two active sites. [10, 12, 13, 45, 46] Unfolded or misfolded proteins tend to expose more hydrophobic residues than do proteins in their native fold. [47]
The present invention is directed to small molecule catalysts of disulfide bond formation and isomerization having improvements over art known catalysts.